Transgenic tomato lines containing Ds elements at defined genomic positions as tools for targeted transposon tagging.

We have introduced a genetically marked Dissociation transposable element (DsHPT) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. Probes for the flanking regions of the T-DNA and transposed DsHPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses. The RFLP map location of 11 T-DNAs carrying DsHPT was determined. The T-DNAs are distributed on 7 of the 12 tomato chromosomes. To explore the feasibility of gene tagging strategies in tomato using DsHPT, we examined the genomic distribution of DsHPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites. After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers beta-glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed DsHPT elements. RFLP mapping of 21 transposed DsHPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.

The mutation bronze-mutable 4 derivative 6856 in maize is caused by the insertion of a novel 6.7-kilobase pair transposon in the untranslated leader region of the bronze-1 gene.

The Ds-controlled allele, bz-m4 Derivative 6856 [bz-m4 D6856], is reported to have an altered temporal- and tissue-specific pattern of gene expression. We have cloned this allele and have characterized it at the molecular level. The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site. The insert is 6.7-kbp long. Ds elements, each approximately 2 kbp in length, are at both ends of the insert. The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene. These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.

Two mutations in a maize bronze-1 allele caused by transposable elements of the Ac-Ds family alter the quantity and quality of the gene product.

The Dissociation (Ds) mutant, Bz-wm, of the maize bronze-1 (bz) locus conditions a leaky phenotype. Plants carrying this mutant allele synthesize a low amount of an altered Bz gene product, which leads to reduced anthocyanin pigmentation in the seed. The molecular analysis reported here shows that the Bz-wm mutant has a 406-bp Ds1 insertion located 63 bp 5' to the start of Bz transcription. Furthermore, the Bz-wm allele contains three additional base pairs within the second exon, relative to the wild-type Bz allele. These additional nucleotides are believed to be derived from the 8-bp target site duplication created by an Activator (Ac) element in a previous allele in the series. The biochemical and molecular analyses of Bz-wm and revertants of Bz-wm indicate that the three additional nucleotides are responsible for the altered enzyme stability, while the Ds1 element affects the steady-state level of Bz-specific protein and RNA. Since the two mutations present in the Bz-wm mutant were each caused by the action of the Ac-Ds transposable element system, these results provide new insights into the ways that transposable elements can modify maize gene expression.

Sequence comparisons of three wild-type Bronze-1 alleles from Zea mays.

We have sequenced genomic clones of two wild-type Bronze-1 (Bz1) alleles, and a cDNA clone from a third wild-type Bz1 allele from maize. Two overlapping transcripts initiate at least 250 bp apart. The first AUG codon after the shorter and more abundant transcript cap site(s) begins the longest open reading frame. The transcript is preceded by a putative TATA box, but not a recognizable CAAT box. The bz1 gene contains a single intron, and exhibits a strong bias for codons with the highest G+C content. Sequence polymorphisms among the Bz1 alleles include deletions/additions, a transposable element insertion, and single base pair substitutions.

RNA splicing permits expression of a maize gene with a defective Suppressor-mutator transposable element insertion in an exon.

The bz-m13CS9 allele of the bronze-1 gene in maize contains a 902-base-pair defective Suppressor-mutator (dSpm) transposable element in the second exon. Nevertheless, 40-50% of the enzymatic activity conditioned by a nonmutant allele at the bronze-1 locus is routinely recovered in crude extracts prepared from plants carrying bz-m13CS9 in the absence of an autonomous Suppressor-mutator element. Analyses of RNAs produced by such plants show that transcription proceeds through the dSpm. The dSpm sequence of the messenger RNA precursor is then removed by RNA splicing using the donor site of the single bronze-1 intron and an acceptor site within the inverted terminal repeat of the dSpm. This results in a messenger RNA with the proper reading frame that could produce a functional enzyme. These data demonstrate that this dSpm insertion in an exon of a structural gene has produced a functional allele with a novel intron consisting, in part, of the dSpm. This mechanism appears to allow dSpm elements to reduce the impact of their insertions on gene expression.

Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable controlling element Activator (Ac).

The bronze (bz) locus of maize has been cloned by an indirect procedure utilizing the cloned transposable controlling element Activator (Ac). Restriction endonuclease fragments of maize DNA were cloned in bacteriophage lambda and recombinant phage with homology to the center of the Ac element were isolated. The cloned fragments were analyzed to determine which contained sequences that were structurally identical to a previously isolated Ac element. Two such fragments were identified. Sequences flanking the Ac element were subcloned and used to probe genomic DNA from plants with well-defined mutations at the bz locus. By this means, it was established that one of the genomic clones contained a bz locus sequence. The subcloned probe fragment was then used to clone a nonmutant Bz allele of the locus. The method described here should prove useful in cloning other loci with Ac insertion mutations.